PROJECT SUMMARY/ABSTRACT We work on molecular mechanisms of nucleotide excision repair and the mammalian circadian clock. We have recently made both the technological and mechanistic progress in both of these areas and obtained direct evidence of inter-connectedness of these two fields. Our findings in both fields are directly applicable to human health. We will apply these new advances for the following objectives: (1) We have developed higher resolution versions of our original XR-seq method for genome-wide single- nucleotide resolution mapping of repair of all DNA damage that is processed by nucleotide excision repair, including damage induced by carcinogens and chemotherapeutic drugs. In addition, we developed Damage-seq methods for similarly genome-wide single nucleotide resolution mapping of DNA damage. We have used the combination of the two methods to discover repair hotspots and coldspots that do not overlap damage hotspots or coldspots and have gained novel information on genome 3D and repair. We will continue characterizing these features to link repair to epigenomic markers, 3D genome organization, chromatin states, and replication timing. XR-seq has also enabled us to discover a novel transcription-coupled repair mechanism in Drosophila and other insects in the order Diptera. We will use biochemical and genetic approaches to solve the mechanism of this novel repair system. (2) We will define the molecular mechanism of the mammalian circadian clock. Recently, we demonstrated that Cryptochrome (CRY), and not Period (PER), is the repressor in the mammalian transcription-translation feedback loop (TTFL), and that PER acts either as a repressor or an activator, depending on the particular gene, in a CRY-dependent manner. We will carry out experiments to reconstitute this model in an in vitro system with purified proteins. (3) Circadian clock, cancer, and chemotherapy. For the first time, we have been able to map both damage formation by cisplatin and its repair in mouse tissues including liver, kidney, and lung, genome-wide and at single- nucleotide resolution. We made the exciting discovery that for most genes the transcribed strand (TS) and non- transcribed strand (NTS) are repaired at different times of the day. We plan to take advantage of this finding to develop more efficient chronotherapy regimens, first for colorectal cancers, and in the future for other types of cancers that are treated with cisplatin and oxaliplatin. The proposed research is innovative because it is based on our discoveries in the fields of DNA repair and circadian clock, and it is significant because of its relevance for cancer prevention and treatment.